Immunohistochemistry was performed using the following monoclonal antibodies against PD‐L1: 22C3 (Dako, Glostrup, Denmark), E1L3N (Cell Signaling Technology, Danvers, MA, USA), and SP263 (Ventana, Tucson, AZ, USA). Staining with clone 22C3 was performed with the corresponding Dako pharmDx assay. Staining with clone SP263 on the Ventana Benchmark Ultra platform was performed with the SP263 Ventana assay. In this study, a score of 50% was defined as “PD‐L1‐high” and a score of less than 50% or equal to 50% as “PD‐L1‐low”. Details of PD‐L1 immunohistochemistry profiles are described in Table S1 .
Sp263
Manufactured by Roche
Sourced in United States, Switzerland
The SP263 is a laboratory equipment product manufactured by Roche. It is designed for use in scientific research and analysis. The core function of the SP263 is to perform specific laboratory tasks, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
E1l3n, by Cell Signaling Technology (14 mentions)
Sp142, by Roche (14 mentions)
Benchmark ultra, by Roche (5 mentions)
Optiview dab ihc detection kit, by Roche (3 mentions)
Ventana benchmark xt, by Roche (3 mentions)
Benchmark ultra platform, by Roche (2 mentions)
Bluing reagent, by Roche (2 mentions)
Benchmark xt, by Roche (2 mentions)
Ultraview universal dab detection kit, by Roche (2 mentions)
Hematoxylin 2, by Roche (2 mentions)
Benchmark, by Roche (2 mentions)
E1l3n, by Roche (2 mentions)
Sk005, by Agilent Technologies (2 mentions)
Dako autostainer link 48 instrument, by Agilent Technologies (2 mentions)
Sk006, by Agilent Technologies (2 mentions)
Bond rx, by Leica Microsystems (2 mentions)
Epr4877, by Abcam (2 mentions)
Bond max, by Leica Biosystems (2 mentions)
Cell conditioning 1 cc1, by Roche (2 mentions)
Ventana optiview dab universal kit, by Roche (2 mentions)
70 protocols using sp263
1
PD-L1 Immunohistochemistry for Cancer
2
PD-L1 Expression in COVID-19 Bronchial Aspirate
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Formalin-fixed paraffin-embedded (FFPE) specimen of bronchial aspirate was obtained from patient # COVID-22 followed at “San Giovanni di Dio e Ruggi D’Aragona” University Hospital. The patient has consented for tissue acquisition per institutional review board-approved protocol. The patient signed informed consent. FFPE tissue sections (4 μm) from the bronchial aspirate sample were used as substrates in immunohistochemical (IHC) reactions. The PD-L1-specific monoclonal antibody (mAb) SP263 and the rabbit mAb IgG, utilized as an isotype control for PD-L1 staining, were purchased from VENTANA. The staining with SP263 mAb was performed on Ventana BenchMark XT automated IHC Stainer (VENTANA) utilizing OptiView DAB IHC Detection Kit (VENTANA). The staining intensity and percentage of stained cells were reviewed and enumerated by an experienced pathologist (PZ) who did not know the patient characteristics and clinical outcome. Staining with PD-L1-specific mAb was performed according to the manufacturers’ instructions and was scored by counting both the number of epithelial and immune stained cells in four high-powered fields.
3
Immunohistochemical Analysis of p53 and PD-L1
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We measured p53 levels by IHC using mouse monoclonal anti-human p53 protein antibody (DO7; Nichirei Biosciences Inc., Tokyo, Japan) and rabbit monoclonal anti-human PD-L1 protein antibody (SP263; Ventana Medical Systems, Inc., Tucson, AZ, USA). Five-micron-thick sections were obtained from formalin-fixed, paraffin-embedded tissues and set aside for p53 antibody (DO7) and PD-L1 (SP263) assay using a VENTANA OptiView DAB universal kit (Roche, Bazel, Switzerland) and VENTANA BenchMark ULTRA automated slide stainer (Roche, Bazel, Switzerland). Heat-induced antigen retrieval was performed using Cell Conditioning 1 (CC1; Ventana Medical Systems) for 32 min at 100 °C, followed by application of the primary antibody against p53 for 16 min at 36 °C, that of CC1 for 64 min at 100 °C, and of the primary antibody against PD-L1 for 16 min at 36 °C.
The IHC results were scored based on the percent positivity of staining. Protein expression of p53 and PD-L1 was evaluated by two pathologists as the percentage of staining area of all tumour cells. p53 status was determined by the percentage range of stained tumour cell nuclei. PD-L1 status was determined by the percentage of tumour cells with membrane staining above background.
The IHC results were scored based on the percent positivity of staining. Protein expression of p53 and PD-L1 was evaluated by two pathologists as the percentage of staining area of all tumour cells. p53 status was determined by the percentage range of stained tumour cell nuclei. PD-L1 status was determined by the percentage of tumour cells with membrane staining above background.
4
Multisite Evaluation of PD-L1 Assays
5
Multisite Evaluation of PD-L1 Assays
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PD-L1 expression was evaluated by quantitative immunofluorescence (QIF) and chromogenic IHC using five monoclonal antibodies (Suppl. Table 1 ), including both LDT and FDA-approved assays. For QIF, clones E1L3N (#13684, Cell Signaling Technology, Inc.), SP142 (#M4420, SpringBio), and SP263 (#790–4905, Ventana Medical Systems, Inc.) were assessed. For chromogenic IHC, automated systems were used for different clones using our own protocol for the LDT E1L3N (#13684, Cell Signaling Technology, Inc.) on multiple platforms, and protocols specified by corresponding manufacturer per the FDA labeling for 22C3 (#SK006, Dako) and 28–8 (#SK005, Dako) with the Dako Autostainer Link 48 Instrument (Dako). Similarly, on label protocols were used for FDA-approved assays; SP263 (#740–4907) and SP142 (#740–4859) both from Ventana Medical Systems, Inc. on the Benchmark Ultra (Ventana Medical Systems, Inc.). For the multi-institutional comparison, twelve 5-µm sections per PD-L1 assay were cut from a block of Index TMA at Yale University and sent to 12 institutions for staining weekly during 6 consecutive weeks, running 2 slides per week with their clinical workload using the assay of choice for each institution.
6
Standardization of PD-L1 Immunostaining Assays
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For the standardization/training set, we used 5 PD-L1 antibodies and 3 automated immunostaining platforms (Leica Bond III, Ventana Benchmark Ultra, and Dako AS Link 48), thus producing 8 combinations of antibodies and platforms. Four standardized PD-L1 assays were performed: 22C3 (Agilent/Dako, PD-L1 pharmDx assay tested in 2 centers), 28-8 (Agilent/Dako, PD-L1 pharmDx assay tested in 2 centers), SP142 (Ventana, Tucson, AZ, USA), and SP263 (Ventana). Tests were performed in accordance with the manufacturer's protocols. LDTs with clone QR1 (Quartett, Berlin, Germany) were tested on the 3 automated immunostaining platforms; LDTs with clone 22C3 were performed on a Leica Bond platform. These LDTs were previously validated in non-small cell lung cancer (21) .
For the validation set of 40 cases, we selected one protocol on each platform based on the best reproducibility (QR1 Leica Bond, SP263 Ventana, and 28-8 Dako).
For the validation set of 40 cases, we selected one protocol on each platform based on the best reproducibility (QR1 Leica Bond, SP263 Ventana, and 28-8 Dako).
7
Comprehensive Molecular and Immune Profiling of Solid Tumors
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PD-L1 status was determined through immunohistochemistry (antibody clone 22C3; Dako or SP263; Ventana) in tumour tissue specimens obtained at initial diagnosis or surgery prior to treatment. The tumour was then assessed by a pathologist for the tumour proportion score (TPS) and/or the combined positive score (CPS). Molecular testing was performed by next-generation sequencing (NGS) or the BRAF V600E real-time PCR assay. The NGS assay (Somatic Solid Tumour Panel, or SSTP) was a customised amplicon-based assay that interrogates mutational hotspots and targeted regions in 26 genes including BRAF, KRAS, NRAS, PIK3CA and TP53. The real-time PCR assay is a TaqMan probe-based allele-specific real-time PCR assay that was modified from the protocol by Benlloch et al. (27 (link)), and tests only for the BRAF c.1799T>A, p.V600E mutation. No screen for fusions was performed in this case series.
For systemic immune profiling, we applied mass cytometry (cytometry by time of flight, CyTOF) to deeply characterise CD8 and CD4 T cells ex vivo, as described previously (28 (link)). Details of the PD-L1 immunohistochemistry, NGS, real-time PCR assay and immune profiling methods are provided in the Supplementary Appendix (see section onsupplementary materials given at the end of this article).
For systemic immune profiling, we applied mass cytometry (cytometry by time of flight, CyTOF) to deeply characterise CD8 and CD4 T cells ex vivo, as described previously (28 (link)). Details of the PD-L1 immunohistochemistry, NGS, real-time PCR assay and immune profiling methods are provided in the Supplementary Appendix (see section on
8
Comprehensive Immunohistochemical Profiling
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Immunohistochemical staining was performed using an automated immunostainer (Benchmark ULTRA, Ventana). The antibodies against the following proteins were: SMARCA2 (EPR23103-44, Abcam, 1:400), ARID1A (EPR13501, Abcam, 1:1000), ARID1B (2D2, Abcam, 1:500), BRG1 (E8V5B, Origene, Ready-to-use), INI1 (25, Origene, Ready-to-use), Napsin-A (IP64, Origene, Ready-to-use), SOX-2 (EP103, Origene, Ready-to-use), P53 (DO-7, Ibp, Ready-to-use), TTF-1 (SPT24, Ibp, Ready-to-use), Ki-67(MyM1-Ki67, Ibp, Ready-to-use), ALK (D5F3, Ventana, Roche), and PD-L1 (SP263, Ventana, Roche).
9
Immunohistochemical Evaluation of SNX20 and PD-L1 in Lung Adenocarcinoma
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Immunohistochemistry assays were performed and quantified as previously described.20 (link) IHC staining was carried out using anti-SNX20 antibody (ab193191) at 1/100 dilution and SP263 (Ventana) assays to test PD-L1 expression for patients with lung adenocarcinoma. The proportion of positively stained cancer cells was graded as follows: 0 (0%), 1 (<10%), 2 (<50%), 3 (<75%), and 4 (≥75%). The staining intensity of the cancer cells was recorded as follows: 0 (no staining), 1 (weak, light yellow), 2 (moderate, yellow brown), and 3 (strong, brown). The staining index (SI) was calculated as the proportion of positively stained cells × the staining intensity, which were displayed as scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12.21 (link) The cutoff values for SNX20 and PD-L1 were determined by receiver operating curve (ROC) analysis. We enlisted the help of two senior pathologists to read the slides for H&E, SNX20, and PD-L1.
10
Measuring PD-L1 Expression in Tumor Tissues
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PD-L1 expression was assessed by conducting an IHC staining of achieved tumor tissues before systemic treatment, using the E1L3N (Cell Signaling Technology, Danvers, MA, USA), 22C3 (Agilent Technologies, Santa Clara, CA, USA), and SP263 (Ventana Benchmark Ultra, Tuscon, AZ, USA) assays. PD-L1-positive tumor cells were considered if the viable tumor cells exhibited any perceptible, partial or complete, membranous or cytoplasmic staining, as previously described [18 (link)]. PD-L1-positive status was defined based on a 1% threshold in immunostained tumor cells in the entire tumor section by any IHC method. The frequency of use of the E1L3N, 22C3, and SP263 IHC assays was as follows: n = 33 (57.9%), 27 (47.4%), and 9 (15.8%), respectively. PD-L1 expression was categorized into three subgroups based on the proportion of immunostained tumor cells using additional cutoff values of 5% and 50%.
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